Human monocytic ehrlichiosis is an emerging tick-borne zoonosis caused by Ehrlichia chaffeensis (Ech), an obligatory intracellular bacterium. The essential virulence factor of Ech is its entry into human monocytes through the route permissive for its replication. To advance our basic understanding and permit development of new therapies, our long-term goal is to elucidate the mechanisms that mediate Ech infectious entry. Our recent study revealed that Ech uses EtpE (Entry triggering protein of Ehrlichia), a unique surface-exposed outer membrane protein to engage the human cell surface glycosylphosphatidyl-inositol-anchored protein DNase X, which induces its entry into host cells to establish prolific infection. How EtpE binding to DNase X triggers infectious Ech entry is unknown. Our preliminary data revealed CD147, hnRNP-K, and N-WASP interact with the EtpE-DNase X complex. Thus, our hypothesis is that EtpE-DNase X binding anchors and activates N- WASP via CD147 and hnRNP-K to elicit spatio-temporal actin polymerization that drives Ech entry. In this application, we will pursue two aims to understand how EtpE binding to DNase X drives infectious Ech entry. Specific Aim 1 is to identify the EtpE-interacting host proteins and their mechanistic contributions to EtpE- triggered Ech entry and infection. We will analyze 1) whether CD147 and hnRNP-K are required for EtpE- coated bead entry, and Ech entry and infection of host cells and mice including use of mice with conditional Cre/LoxP CD147 knock-out in macrophages; 2) the spatio-temporal recruitment profiles of CD147 and hnRNP- K at entry foci of Ech and of EtpE-coated beads, by live-cell/intravital fluorescence microscopy; 3) proteins involved in the EtpE-host protein interaction, using a) tandem affinity purification and b subtractive multidimensional protein identification technology; 4) the determinants/kinetics of the binding of CD147 and hnRNP-K to the EtpE-C-DNase X complex, by fluorescence resonance transfer, in vivo bimolecular fluorescence complementation, and in vitro binding assays, using defined domains and mutant proteins; and 5) monocyte tropism using human neutrophils and a granulocyte-tropic obligatory intracellular bacterium. Specific Aim 2 is to elucidate mechanisms to coordinate the spatio-temporal N-WASP activation at Ech entry foci. We will analyze 1) recruitment of N-WASP at entry foci of Ech and of EtpE-coated beads in a DNase X, CD147, and hnRNP-K-dependent manner; 2) requirement of N-WASP for entry of Ech and EtpE-coated beads, and infection of host cells and mice including the use of conditional Cre/LoxP N-WASP knock-out in macrophages; 3) requirement of DNase X, CD147, hnRNP-K, and N-WASP for EtpE-triggered polymerization of pyrenyl-actin in vitro; and 4) the spatio-temporal recruitment profiles of N-WASP and F-actin to entry foci of Ech and EtpE- coated beads, using live-cell imaging. If our hypothesis is proven, EtpE would be the first bacterial molecule described to activate N-WASP through a unique protein complex for infectious entry. The identified pathways will provide a platform for developing new prophylactic and therapeutic strategies against Ech.